1. Field of the Invention
This invention relates to a method for isolating and culturing Schwann cells, to the isolated Schwann cells per se and to uses thereof. In particular, the invention provides a method for enhancing the survival and proliferation of human Schwann cells by culturing them in serum free culture medium supplemented with a Rse/Axl receptor activator and other mitogenic agents.
2. Description of Related Art
1. Schwann cells
Schwann cells are the principal support cells in the peripheral nervous system. The cells originate from the neural crest during early embryonic development and migrate with the extending axons of the nerve into the periphery. During this phase, Schwann cells undergo rapid proliferation to produce an adequate number of cells to accommodate the growing axons. Subsequently, Schwann cells become terminally differentiated by ensheathing or myelinating the axons and then remain quiescent during adult life. However, Schwann cell proliferation can be stimulated under pathological conditions and plays a crucial role in nerve regeneration following injury. When a peripheral nerve is transected, Schwann cells at the site of the injury begin to demyelinate and re-enter the cell cycle (Bunge, Current Opin. in Neurobiol. 3:805 [1993]). The proliferating Schwann cells produce neurotropic factors and extracellular matrix proteins to guide or facilitate the regrowth of the transected axons and finally complete the process of regeneration by remyelinating the regenerated axons.
The remarkable capacity of Schwann cells to promote nerve fiber regeneration, in both the peripheral and central nervous system, has been demonstrated by peripheral nerve graft (Paino and Bunge, Experimental Neurology 114:254 [1991]) and the implantation of guidance channels impregnated with Schwann cells (Guenard et al., J. Neuroscience 12(9):3310 [1992] and Paino et al., J. Neurocytol. 23:433 [1994]). A cellular prosthesis containing human Schwann cells has been proposed for clinical applications such as transplantation to the site of spinal cord injury to influence the regeneration of central axons and to repair complex peripheral nerve injuries containing lengthy gaps (Levi et al., J. Neuroscience 14(3):1309 [1994]). The clinical success of these procedures, which use autologous Schwann cells, depends on the ability to expand in vitro a pure Schwann cell population starting from material in a small biopsy.
Several reports have described techniques for culturing rat Schwann cells. See Brockes et al., Brain Research 165:105-118 (1979); Brockes et al., J. Biol. Chem. 255(18):8374-8377 (1980); Brockes et al., Ann. Neurol. 20(3):317-322 (1986); Brockes, J., Methods in Enzym. 147:217-225 (1987); Morrissey et al., J. Neuroscience 11(8):2433-2442 (1991); Paino and Bunge, Experimental Neurology 114:254 (1991); Guenard et al., J. Neuroscience 12(9):3310-3320 (1992); Peulve et al., Exper. Cell Res. 214:543-550 (1994); Li et al., J. Neuroscience 14(7):4050-4063 (1994); Collier and Martin, Exper. Neurol. 124.129-133 (1993); Scherer et al., J. Neuroscience Res. 38:575-589 (1994); Yamamoto et al., Brain Res. 653:335-339 (1994); Morgan et al. Development 120:1399-1409 (1994); Paino et al. J. Neurocytol. 23:433-452 (1994); Messing et al., J. Neurosci. 14:3533-3539 (1994); Haynes et al., J. Neurosci. Methods 52:119-127 (1994); WO 92/03536; WO 92/18627; WO 94/00140; and WO 94/04560.
Watabe et al., J. Neurosci. Res. 39:525-534 (1994) studied the mitogenic effects of platelet-derived growth factor, fibroblast growth factor, transforming growth factor-.beta., and heparin-binding serum factor on adult mouse Schwann cells in cell culture.
Recently, techniques for culturing human Schwann cells have been described. See Rutkowski et al., Ann. Neurol. 31(6):580-586 (1992); Levi et al., J. Neuroscience 14(3):1309-1319 (1994); and Levi et al., J. Neuroscience 15(2):1329-1340 (1995).
Traditionally, Schwann cells have been grown in culture medium supplemented with serum. Fibroblasts are a major contaminant of these preparations, particularly when adult tissues are used, and will overgrow the Schwann cells. The fibroblast population varies with the time in the culture (Levi et al., J. Neuroscience 14(3):1309 [1994]), or is reduced through laborious protocols such as sequential outgrowth (Morrissey et al., J. Neuroscience 11(8):2433-2442 [1991]), antibody selection (Brockes, J., Methods in Enzym. 147:217-225 [1987] and Watabe et al., J. Neurosci. Res. 39:525-534 [1994]) or use of anti-mitotic agents (Levi et al., J. Neuroscience 15(2):1329-1340 [1995]).
2. Rse and Axl receptors
Mark et al. recently described the human and murine complementary DNA sequences of the receptor tyrosine kinase Rse that is preferentially expressed in the adult brain (Mark et al., J. Biol. Chem. 269:10720 [1994]). The extracellular domain of Rse receptor is composed of two immunoglobulin-like (Ig-L) repeats followed by two fibronectin type III repeats. Complementary DNA sequences encoding proteins identical to human (Ohashi et al., Oncogene 9:699 [1994]) and murine Rse (Lai et al., Oncogene 9:2567 [1994]) have been reported independently, and termed Sky and Tyro3, respectively. See also Fujimoto and Yamamoto Oncogene 9:693 (1994) concerning the murine equivalent to Rse they call brt and Dai et al. Oncogene 9:975 (1994) with respect to the human molecule they call tif.
The expression of Rse in various tissues has been investigated. Lai et al., Oncogene 9:2567 [1994], found that, in the adult brain, Rse mRNA is localized in neurons of the neocortex, cerebellum and hippocampus. Schulz et al. similarly found that Rse is expressed at high levels in the cerebral cortex, the lateral septum, the hippocampus, the olfactory bulb and in the cerebellum. The highest levels of Rse expression in the brain were found to be associated with neurons. (Schulz et al. Molec. Brain Res. 28:273-280 [1995]). In the central nervous system (CNS) of mice, the expression of Rse was detected at highest levels during late embryonic stages and post birth, coincident with the establishment and maintenance of synaptic circuitry in cortical and hippocampal neurons (Lai et al., Oncogene 9:2567 [1994] and Schneider et al., Cell 54:787-793 [1988]). This process is believed to be regulated locally, by cells that are in direct contact or positioned close to one another.
Rse is structurally related to Axl (also known as Ufo or Ark) and shares 43% overall amino acid sequence identity with this tyrosine kinase receptor. See O'Bryan et al., Mol. Cell. Biol. 11:5016 (1991), Janssen et al., Oncogene 6:2113 (1991), Rescigno et al. Oncogene 5:1908 (1991) and Bellosta et al. 15:614 (1995) concerning Axl. Rse and Axl, together with c-Mer (Graham et al., Cell Growth Differ. 5:647 [1994]), define a class of receptor tyrosine kinases whose extracellular domains resemble neural cell recognition and adhesion molecules (reviewed by Ruitishauser, U. in Current Opin. Neurobiology 3:709 [1993] and Brummendorf and Rathjen in J. Neurochemistry 61:1207 [1993]). Like Rse, Axl is also expressed in the nervous system, but is more widely expressed than Rse in peripheral tissues.
Putative ligands for the Rse and Axl receptors have been reported. Varnum et al. Nature 373:623 (1995) and Stitt et al., Cell 80:661-670 (1995) recently reported that gas6 (for growth arrest-specific gene 6) is a ligand for Axl. Gas6 belongs to a set of murine genes which are highly expressed during serum starvation in NIH 3T3 cells (Schneider et al., Cell 54:787-793 [1988]). These genes were designated growth arrest-specific genes, since their expression is negatively regulated during growth induction. The human homolog of murine gas6 was also cloned and sequenced by Manfioletti et al. in Molec. Cell Biol. 13(8):4976-4985 (1993). They concluded that gas6 is a vitamin K-dependent protein and speculated that it may play a role in the regulation of a protease cascade relevant in growth regulation. Gas6 is expressed in a variety of tissues including the brain. See also Colombo et al. Genome 2:130-134 (1992) and Ferrero et al. J. Cellular Physiol. 158:263-269 (1994) concerning gas6.
Stitt et al., Cell 80:661-670 (1995) further reported that protein S is the ligand for Tyro3. Protein S is a vitamin K-dependent plasma protein that functions as an anticoagulant by acting as a cofactor to stimulate the proteolytic inactivation of factors Va and VIIIa by activated protein C. Reviewed in Esmon et al. Aterioscler. Thromb. 12:135 (1992). Accordingly, protein S is an important negative regulator of the blood-clotting cascade. See Walker et al., J. Biol. Chem. 255:5521-5524 (1980), Walker et al., J. Biol. Chem. 256:11128-11131 (1981), Walker et al., Arch. Biochem. Biophys. 252:322-328 (1991), Griffin et al. Blood 79:3203 (1992) and Easmon, D., Aterioscler. Thromb. 12:135 (1992). The discovery that about half of the protein S in human plasma is bound to C4BP further supports the notion that protein S is involved in the complement cascade. Dahlback et al., PNAS(USA) 78:2512-2516 (1981). The role of protein S as a mitogen for smooth muscle cells has also been reported. Gasic et al., PNAS (USA) 89:2317-2320 (1992).
Protein S can be divided into four domains (see FIGS. 1A, 1C and 1D herein). Residues 1-52 (Region A) are rich in .gamma.-carboxyglutamic acid (Gla) residues which mediate the Ca.sup.2+ dependent binding of protein S to negatively charged phospholipids (Walker, J. Biol. Chem. 259:10335 [1984]). Region B includes a thrombin-sensitive loop. Region C contains four epidermal growth factor (EGF)-like repeats. Region D is homologous to the steroid hormone binding globulin (SHBG) protein (Hammond et al., FEBS Lett. 215:100 [1987]). As discussed by Joseph and Baker (FASEB J. 6:2477 [1994]), this region is homologous to domains in the A chain of laminin (23% identity) and merosin (22% identity) and to a domain in the Drosophila crumbs (19%).
Murine and human gas6 cDNAs encode proteins having 43 and 44% amino acid sequence identity respectively to human protein S.